細(xì)胞周期素依賴性蛋白激酶調(diào)控細(xì)胞死亡的研究
發(fā)布時(shí)間：2019-06-20 00:02
【摘要】： 第一部分 CDK1與細(xì)胞凋亡 目的：探討CDK1在細(xì)胞凋亡中的重要作用及其機(jī)制研究。 材料和方法：先后構(gòu)建了表達(dá)CDK1的質(zhì)粒、突變了CDK1主要磷酸化位點(diǎn)(包括Thr161和Tyr15)的質(zhì)粒和能有效沉默CDK1表達(dá)的RNAi，以肝癌細(xì)胞Hela為研究對(duì)象，以紫外打擊為凋亡的主要誘導(dǎo)方式，采用MTT法、流式細(xì)胞學(xué)分析、流式細(xì)胞學(xué)分選技術(shù)、免疫熒光分析、蛋白質(zhì)免疫共沉淀技術(shù)、Western blot分析等技術(shù)分析細(xì)胞在遭受外界打擊的條件下，CDK1蛋白與凋亡相關(guān)蛋白的相互作用關(guān)系。 結(jié)果： 1)我們成功構(gòu)建了能夠表達(dá)CDK1蛋白的真核表達(dá)質(zhì)粒，能夠有效沉默CDK1表達(dá)的RNAi和突變了Thr161位蛋白和Tyr15位蛋白的表達(dá)CDK1質(zhì)粒；高表達(dá)CDK1的細(xì)胞在紫外線打擊下凋亡率顯著增加；沉默CDK1表達(dá)的細(xì)胞在紫外線打擊下凋亡率顯著下降；進(jìn)而發(fā)現(xiàn)，當(dāng)細(xì)胞引入突變了Tyr15位蛋白的CDK1質(zhì)粒時(shí)，紫外誘導(dǎo)的G1期特異性細(xì)胞凋亡是增加的；當(dāng)細(xì)胞引入突變了Thr161位蛋白的CDK1質(zhì)粒時(shí)，紫外打擊導(dǎo)致的細(xì)胞的G1期凋亡相對(duì)有所減少，免疫共沉淀顯示帶有Flag尾Tyr15突變體其Thr161位點(diǎn)可以被磷酸化。 2)在低血清培養(yǎng)誘導(dǎo)的細(xì)胞G1期阻滯模型中，，當(dāng)紫外打擊后根據(jù)不同的時(shí)間點(diǎn)我們對(duì)細(xì)胞進(jìn)行了分選，與對(duì)照組相比，紫外打擊組細(xì)胞內(nèi)Phospho-cdc2(Tyr15)的表達(dá)是逐漸下降，而同時(shí)出現(xiàn)了Phospho-cdc2(Thr161)表達(dá)的上升；對(duì)照組和紫外打擊組內(nèi)CDK2處于低水平表達(dá)，沒(méi)有出現(xiàn)顯著性改變；凋亡相關(guān)蛋白檢測(cè)發(fā)現(xiàn)抗凋亡蛋白Bc12的表達(dá)上調(diào)。 3)對(duì)細(xì)胞紫外線打擊后分別提取胞核蛋白和胞漿蛋白，結(jié)果顯示紫外打擊組胞核中Phospho-cdc2(Tyr15)表達(dá)與對(duì)照組無(wú)明顯差異，而胞漿中出現(xiàn)了顯著下降。 4)免疫熒光分析顯示，細(xì)胞在紫外打擊后少量的Phospho-cdc2(Tyr15)出現(xiàn)了聚集于凋亡小體處的現(xiàn)象，Phospho-cdc2(Thr161)濃聚在胞漿中。 結(jié)論： 1)CDK1表達(dá)過(guò)多可以促使凋亡的發(fā)生，抑制CDK1的表達(dá)可以減少細(xì)胞的凋亡。 2)通過(guò)向細(xì)胞引入外源性表達(dá)的CDK1相關(guān)磷酸化位點(diǎn)突變體后其Thr161蛋白可以被磷酸化，而磷酸化的后果是促發(fā)了凋亡。而引入Thr161突變的CDK1并沒(méi)有顯著抑制凋亡的發(fā)生。 3)從時(shí)間的觀點(diǎn)看來(lái)，CDK1在G1期如果出現(xiàn)了異常激活則可能直接導(dǎo)致凋亡的發(fā)生；從空間的角度理解，CDK1 Thr161磷酸化后若異常停留在胞漿中，和(或)CDK1Tyr15以磷酸化的方式入核或在胞漿中去磷酸化也是凋亡發(fā)生的必然條件之一。 第二部分 CDK1與細(xì)胞自噬 目的： 探討CDK1在細(xì)胞自噬中的重要作用。 材料和方法： 先后構(gòu)建了高表達(dá)CDK1的質(zhì)粒和能有效沉默CDK1表達(dá)的RNAi，以肝癌細(xì)胞Hela為研究對(duì)象，通過(guò)DHanks液饑餓為主要誘導(dǎo)方式，采用流式細(xì)胞學(xué)分析、免疫熒光分析、電鏡和Western blot分析等技術(shù)分析細(xì)胞在饑餓所誘導(dǎo)的自噬中，CDK1蛋白在其中的作用。 結(jié)果： 當(dāng)細(xì)胞導(dǎo)入高表達(dá)CDK1后，我們可以見(jiàn)到相對(duì)于對(duì)照組，其自噬相關(guān)蛋白表達(dá)增加，自噬泡更多且更大；相反，當(dāng)我們沉默CDK1的表達(dá)后，細(xì)胞自噬的現(xiàn)象明顯受到抑制。 結(jié)論： CDK1表達(dá)過(guò)多可以促使細(xì)胞自噬的發(fā)生，抑制CDK1的表達(dá)可以減少細(xì)胞的自噬。
[Abstract]:the first part CDK1 vs. The purpose of cell apoptosis: to study the important role of CDK1 in the apoptosis of cells Materials and methods: The expression of the plasmid of CDK1, the plasmid of the main phosphorylation site of CDK1 (including Thr161 and Tyr15) and the RNAi which can effectively silence the expression of CDK1 were constructed. The methods of induction, MTT, flow cytometry, flow cytometry, immunofluorescence, protein-immunoprecipitation and Western blot were used to analyze the CDK1 protein and apoptosis. related egg Results:1) We successfully constructed the eukaryotic expression plasmid which can express the CDK1 protein, can effectively silence the RNAi of the CDK1 expression and the expression of the expression CDK1 of the Tyr15-bit protein and the high expression CD. The apoptosis rate of the cells of K1 cells increased significantly under the ultraviolet ray; the apoptosis rate of the cells expressed by the silence CDK1 was significantly decreased under the ultraviolet ray; furthermore, when the cells were introduced into the CDK1 plasmid with the mutation of the Tyr15-bit protein, the apoptosis of the UV-induced G1 phase-specific cells was increased; and when the cells were introduced into the process, When the expression of the CDK1 plasmid of the Thr161-bit protein was changed, the apoptosis of the G1 phase of the cells caused by the ultraviolet-attack was relatively reduced, and the immunoprecipitation showed that the Flag tail Tyr1 5. The Thr161 site of the mutant can be phosphorylated.2) In the cell G1 phase block model induced by low serum culture, the cells were sorted according to different time points after UV-attack, and compared with the control group, the expression of Phospho-cdc2 (Tyr15) in the ultraviolet-striking group was gradually decreased, while Ph. Phospho-cdc2 (Thr161) expression increased; in the control group and in the ultraviolet-striking group, the CDK2 was expressed at a low level and no significant change was observed; and The expression of anti-apoptotic protein Bc12 was found to be up-regulated by the detection of apoptosis-related protein. R15) The expression of r15 showed no significant difference with the control group, and there was a significant decrease in the cytoplasm.4) Immunofluorescence analysis showed that the small amount of Phospho-cdc2 (Tyr15) in the cells appeared to be clustered in the apoptotic body after the ultraviolet. the present at the office Phospho-cdc2 (Thr161) is concentrated in the cytoplasm. 1) The expression of CDK1 can induce apoptosis, and the expression of CDK1 can reduce the apoptosis of the cells. The Thr161 protein of the DK1-related phosphorylation site mutant can be phosphorylated And the effect of phosphorylation is to promote apoptosis. CDK1, which is introduced to the Thr161 mutation, does not significantly inhibit the occurrence of apoptosis.3) From the point of view of time, CDK1 is abnormal in G1 phase. Activation may lead to the occurrence of apoptosis directly; from the point of view of the space, after the phosphorylation of CDK1 Thr161, in that slurry, and (or) CDK1Ty r15 to enter the nucleus in a phosphorylated manner or to dephosphorylation in the cytoplasm It is also the occurrence of apoptosis One of the necessary conditions for CDK1 and autophagy: to study the important role of CDK1 in autophagy. Materials and methods: high expression of CDK1 has been constructed. The plasmid and the RNAi which can effectively silence the expression of the CDK1, the liver cancer cell Hela is the research object, is the main In this paper, the role of CDK1 protein in the autophagy induced by starvation was analyzed by flow cytometry, immunofluorescent analysis, electron microscope and Western blot analysis. to be fine After the introduction of high-expression CDK1, we can see the increase of autophagy-related protein expression and autophagy relative to the control group.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329
本文編號(hào)：2502742
[Abstract]:the first part CDK1 vs. The purpose of cell apoptosis: to study the important role of CDK1 in the apoptosis of cells Materials and methods: The expression of the plasmid of CDK1, the plasmid of the main phosphorylation site of CDK1 (including Thr161 and Tyr15) and the RNAi which can effectively silence the expression of CDK1 were constructed. The methods of induction, MTT, flow cytometry, flow cytometry, immunofluorescence, protein-immunoprecipitation and Western blot were used to analyze the CDK1 protein and apoptosis. related egg Results:1) We successfully constructed the eukaryotic expression plasmid which can express the CDK1 protein, can effectively silence the RNAi of the CDK1 expression and the expression of the expression CDK1 of the Tyr15-bit protein and the high expression CD. The apoptosis rate of the cells of K1 cells increased significantly under the ultraviolet ray; the apoptosis rate of the cells expressed by the silence CDK1 was significantly decreased under the ultraviolet ray; furthermore, when the cells were introduced into the CDK1 plasmid with the mutation of the Tyr15-bit protein, the apoptosis of the UV-induced G1 phase-specific cells was increased; and when the cells were introduced into the process, When the expression of the CDK1 plasmid of the Thr161-bit protein was changed, the apoptosis of the G1 phase of the cells caused by the ultraviolet-attack was relatively reduced, and the immunoprecipitation showed that the Flag tail Tyr1 5. The Thr161 site of the mutant can be phosphorylated.2) In the cell G1 phase block model induced by low serum culture, the cells were sorted according to different time points after UV-attack, and compared with the control group, the expression of Phospho-cdc2 (Tyr15) in the ultraviolet-striking group was gradually decreased, while Ph. Phospho-cdc2 (Thr161) expression increased; in the control group and in the ultraviolet-striking group, the CDK2 was expressed at a low level and no significant change was observed; and The expression of anti-apoptotic protein Bc12 was found to be up-regulated by the detection of apoptosis-related protein. R15) The expression of r15 showed no significant difference with the control group, and there was a significant decrease in the cytoplasm.4) Immunofluorescence analysis showed that the small amount of Phospho-cdc2 (Tyr15) in the cells appeared to be clustered in the apoptotic body after the ultraviolet. the present at the office Phospho-cdc2 (Thr161) is concentrated in the cytoplasm. 1) The expression of CDK1 can induce apoptosis, and the expression of CDK1 can reduce the apoptosis of the cells. The Thr161 protein of the DK1-related phosphorylation site mutant can be phosphorylated And the effect of phosphorylation is to promote apoptosis. CDK1, which is introduced to the Thr161 mutation, does not significantly inhibit the occurrence of apoptosis.3) From the point of view of time, CDK1 is abnormal in G1 phase. Activation may lead to the occurrence of apoptosis directly; from the point of view of the space, after the phosphorylation of CDK1 Thr161, in that slurry, and (or) CDK1Ty r15 to enter the nucleus in a phosphorylated manner or to dephosphorylation in the cytoplasm It is also the occurrence of apoptosis One of the necessary conditions for CDK1 and autophagy: to study the important role of CDK1 in autophagy. Materials and methods: high expression of CDK1 has been constructed. The plasmid and the RNAi which can effectively silence the expression of the CDK1, the liver cancer cell Hela is the research object, is the main In this paper, the role of CDK1 protein in the autophagy induced by starvation was analyzed by flow cytometry, immunofluorescent analysis, electron microscope and Western blot analysis. to be fine After the introduction of high-expression CDK1, we can see the increase of autophagy-related protein expression and autophagy relative to the control group.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329
【引證文獻(xiàn)】
相關(guān)期刊論文 前2條
1 李瑞;鄭亞莉;周學(xué)兵;王煒;;Cdk1在HepG2細(xì)胞的表達(dá)及活性對(duì)肝癌細(xì)胞凋亡的影響[J];寧夏醫(yī)科大學(xué)學(xué)報(bào);2011年06期
2 徐紀(jì)偉;孫丹華;;大鼠脊髓半切損傷后細(xì)胞周期蛋白激酶1的表達(dá)增強(qiáng)[J];細(xì)胞與分子免疫學(xué)雜志;2013年10期
相關(guān)碩士學(xué)位論文 前1條
1 李瑞;CDK1在HepG2細(xì)胞的表達(dá)及活性對(duì)肝癌細(xì)胞凋亡的影響[D];寧夏醫(yī)科大學(xué);2011年
本文編號(hào)：2502742
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